Our objective is to develop a Protease Chain Reaction technology (ProCR) that will enable ultra- sensitive and highly-quantitative detection of cancer markers. The basic element of a protease chain reaction is a protease-inhibitor complex. The protease is inactive when bound to the inhibitor but, once freed, is capable of cleaving the inhibitor and releasing additional free protease. A trace of free protease initiates the chain reaction and eventually causes the release of all protease from the inhibitory complex. The lag time preceding the full release of protease is determined by the initial concentration of free protease. The Phase I project developed a first-generation of self-amplifying complexes that form the core of the ProCR detection technology. All Phase I milestones were significantly exceeded. In Phase II we will develop powerful molecular sensors based on self- amplifying complexes. A sensor consists of three elements: 1) a sensing element that responds to the presence of an analyte; 2) the processing/computational element that amplifies and quantifies the signal from the detection element; and 3) the transducer element that produces an optical signal. Through this combination, extremely sophisticated enzymatic sensors will be built to detect cancer markers. Accordingly the three experimental Aims are to: 1) Engineer sensing elements; 2) Engineer enhanced processing/computational elements; and 3) Engineer signaling components. Fundamentally, a protein chain reaction is a powerful analogue computer with two key characteristics that greatly facilitate the detection of target molecules. 1) It can convert th concentration of a specific target molecule into a time signature. 2) It can create enormous signal amplification, analogous to the amplification of DNA by a polymerase chain reaction (PCR). Thus detection is enabled because the final observable signal produced by a target molecule can be very large and the time lag until onset of the signal is precisely correlated with the concentration of target molecule. The long term goal is to develop protease-inhibitor complexes as enzymatic nano-processors which can be combined to detect multiple signals and to control output with multiple logic gates.